CN111718398B - HRas protein-targeted alpha-helix polypeptide inhibitor and application thereof - Google Patents

Abstract

The invention relates to an alpha helical polypeptide inhibitor of a targeted HRas protein, which has the structural formula shown as follows:

the invention also provides application of the polypeptide inhibitor in preparing a medicament for targeting HRas protein. The HRas protein targeted alpha helix polypeptide inhibitor can be applied to radiation sensitizers for radiotherapy.

Description

HRas protein-targeted alpha-helix polypeptide inhibitor and application thereof

Technical Field

The invention belongs to the field of bioengineering, and relates to a polypeptide, in particular to an alpha helix polypeptide inhibitor targeting HRas protein and application thereof.

Background

There are three major therapeutic modalities in the cancer treatment field: radiation therapy, chemotherapy and surgery, radiation therapy being one of the important non-surgical treatments for cancer. The radiotherapy is called as 'invisible scalpel', can keep organs and functions thereof, and achieves the aim of minimally invasive and efficient tumor treatment, for example, for treatment of nasopharyngeal carcinoma, because the nasopharyngeal carcinoma is dissected and located in a special position, and the operation treatment is very difficult, but nasopharyngeal carcinoma cells are sensitive to radioactive rays, the radiotherapy is an ideal treatment mode for treating the nasopharyngeal carcinoma, and early-stage nasopharyngeal carcinoma patients can achieve good cure rate by adopting a radiotherapy strategy. To date, the combined use of radiation therapy and chemotherapy has been widely used in tumor therapy worldwide. However, the radiation resistance produced by tumor cells may impair the effectiveness of radiation therapy. Radiation sensitizers have been shown to be effective in some cancers such as prostate cancer, radiation sensitizers can increase the radiation sensitivity of tumor cells to X-rays and the like, protect normal cells and tissues to the greatest extent possible while killing tumor cells at high efficiency, and various radiation sensitizers such as small molecule drugs and RNA drugs have been developed to enhance antitumor effects. Recently, radiotherapy sensitizers to specific target proteins have attracted a great deal of interest in both academia and industry. Various studies have shown that Ras signaling pathways play an important role in the regulation of radiation resistance. The research shows that rigosetib is a regulator for inhibiting Ras-Raf interaction, can interrupt Ras-Raf-MEK-ERK and PI3K/AKT signal pathway, can be used as a radiosensitizer for cervical cancer radiotherapy, and can inhibit the proliferation of cancer cells through G2/M cell cycle block and other mechanisms. In 2005, Kim et al discovered that Ras-targeting siRNA could down-regulate phosphorylation levels of AKT and ERK kinase (MAPK) levels, reduce cell survival in clonogenic experiments, and show radiosensitization. Therefore, the development of effective inhibitors to block the Ras signaling pathway may be a viable strategy to develop effective radiosensitizers.

Based on the interaction sequence of Ras-Sos interface, Patgiri et al developed an alpha helical polypeptide HBS 3 that targets HRas using a hydrogen bond replacement strategy (HBS), which directly targets HRas protein and successfully down-regulates the phosphorylation cascade downstream of Ras signaling. In 2015, leshciner et al developed an all-carbon "stapled peptide" SAH-SOS1A against wild-type and mutant KRas, which was effective in down-regulating phosphorylation levels of ERK and AKT proteins in the Ras signal downstream phosphorylation cascade and inhibiting growth of cancer cells containing KRas mutant genes. HBS 3 polypeptide and SAH-SOS1A polypeptide are key polypeptide fragments based on Ras-Sos interaction interface, namely, the polypeptide inhibitor of the targeted Ras protein is obtained by simulating the amino acid mutation of alpha H helix (amino acid residue 929-944) on the Sos protein and screening suitable ring-closing positions, and the polypeptide design strategy provides an idea for developing the polypeptide inhibitor of the targeted Ras protein. In 2015, Udadhyaya et al developed a cyclic peptide 9A5 with better cell membrane permeability, which could block Ras-effector interaction and was found in subsequent studies to induce cancer cell apoptosis.

Research has reported that an N-terminal aspartic acid nucleation strategy (TD strategy) is developed by constructing an (i, i +3) lactam bond between 2, 3-diaminopropionic acid (Dap) and L-isoaspartic acid at the N-terminal end of the polypeptide, which can immobilize the polypeptide in an alpha-helical structure, and the polypeptide constructed by this strategy has improved stability of the polypeptide and cell membrane permeability. The polypeptide inhibitor developed at present may have the defects of weak membrane penetrating capability of cell membranes, low binding affinity with HRas and the like, and the development of more efficient Ras inhibitors or Ras-related radiotherapy sensitizers is still needed. The use of N-terminal aspartate nucleation strategy to construct stable alpha helix polypeptides to target Ras proteins is a viable approach.

Methyl auristatin E is called MMAE for short and is a synthetic antitumor drug. MMAE is a potentially potent radiosensitizer of X-ray radiation that increases the sensitivity of tumor cells to radiation, a powerful polypeptide cytotoxin and mitotic suppressive agent that inhibits cell division by blocking tubulin polymerization, derived from a polypeptide known as dolastatin, present in the marine mollusk Dolabella auricularia. MMAE polypeptides and antibody conjugates thereof have shown potent in vitro and in vivo activity against a variety of lymphomas, leukemias and solid tumors in preclinical studies. The MMAE can be used as a positive control for researching a relevant experiment of tumor cells on radiation sensitivity, and meanwhile, in order to verify the potential application prospect of the combination therapy of the MMAE and the polypeptide of the targeted Ras protein, the invention further evaluates the research of the polypeptide for inhibiting the Ras signal pathway and the MMAE combination drug on the aspect of X-ray radiation radiosensitization.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention provides an HRas protein targeted alpha helix polypeptide inhibitor and application thereof, and aims to solve the technical problems that the polypeptide inhibitor in the prior art is weak in cell membrane penetrating capability and low in binding affinity with HRas.

The invention provides an alpha helix polypeptide inhibitor targeting HRas protein, which has a structural formula shown as follows (named as H5 polypeptide):

wherein isoD is L-isoaspartic acid (L-isoaspartic acid), and Dap represents 2, 3-diaminopropionic acid.

Further, the invention also provides application of the HRas protein targeted alpha helix polypeptide inhibitor in preparation of a drug for targeting HRas protein.

Furthermore, the invention also provides application of the HRas protein targeted alpha helix polypeptide inhibitor in preparing a medicament for treating the HRas high-expression cervical cancer cell line.

Furthermore, the invention also provides application of the HRas protein targeted alpha-helix polypeptide inhibitor in preparing a medicament for increasing the sensitivity of cancer cell radiotherapy.

Furthermore, the invention also provides a pharmaceutical composition, which contains the HRas protein targeted alpha-helix polypeptide inhibitor and monomethyl auristatin E, wherein the concentration of the HRas protein targeted alpha-helix polypeptide inhibitor in the composition is 10 mu M, and the concentration of the monomethyl auristatin E is 0.5 nM.

Further, the invention also provides application of the pharmaceutical composition in preparation of a medicament for targeting HRas protein.

Further, the invention also provides application of the pharmaceutical composition in preparing a medicament for treating the HRas high-expression cervical cancer cell line.

Furthermore, the invention also provides application of the pharmaceutical composition in preparing a medicament for increasing the sensitivity of cancer cell radiotherapy.

The invention is based on a key polypeptide sequence on the Sos protein of an interaction pocket in a Ras-Sos compound crystal structure, namely an alpha H helix (amino acid residue 929-944) on the Sos protein, the polypeptide sequence is used as a linear control polypeptide L1 (amino acid residue 929-944: FFGIYLTNILKTEEGN), a series of ring-closing polypeptides are prepared by using an N-terminal aspartic acid nucleation strategy, a fluorescence polarization method is used for detecting the binding affinity and the interaction of a stable polypeptide and the Ras protein, and the killing effect of the polypeptide on cancer cells and the application of the polypeptide in the cancer cell radiotherapy sensitization direction are further evaluated at a cell level.

The invention further discusses the application effect of the combined drug of the polypeptide and the MMAE in the aspect of X-ray radiation sensitization. In the invention, a series of experimental screens are carried out to determine a potential polypeptide H5 which has the advantages of high binding affinity with HRas, strong membrane penetrating capability, good cell activity and the like, and then the killing effect of the drug combination of the polypeptide and MMAE on HeLa cells is further evaluated, and the application of the drug combination of the polypeptide and MMAE on the aspect of radiotherapy sensitization of induced HeLa cells is evaluated by a clone formation experiment.

The invention designs and synthesizes H1-H12 and other polypeptides, the specific polypeptide sequence is shown in Table 1, and the expression mode of the polypeptide sequence is the amino acid sequence from N end to C end. The polypeptide L1 contains the original sequence of the so 1. alpha. H-helix (amino acids 929-944: FFGIYLTNILKTEEGN), arginine Arg with positive charge is synthesized at the end of all the polypeptides, in order to further increase the cell membrane permeability of the polypeptides by increasing the positive charge of the polypeptides, thereby enhancing the biological activity of the polypeptides, and Trp is additionally added at the end of all the polypeptides for the purpose of concentration determination of the polypeptides. The polypeptide H1/H2 is based on a sequence of a polypeptide L1, a nucleation template at the N terminal is utilized to introduce Dap and isoD amino acids into the N terminal of the polypeptide, a stable loop-closing polypeptide is constructed between the Dap and the isoD amino acids in an (i, i +3) amido bond loop-closing mode, and a proper loop-closing position is screened. The polypeptides H3 and H4 were designed based on the optimized polypeptide sequence (FEGIYRLELLKAEEAN) previously reported by Patgiri et al, again with an amide bond loop at a different amino acid position at the N-terminus.

For polypeptides H5, H6 and H7, the amino acid residue glutamic acid Glu at position 941 on the polypeptide sequence was replaced with glutamine Gln to further increase the overall positive charge of the polypeptide to improve cell membrane penetration. Other polypeptides based on fluorescence polarization experiments, non-essential hydrophobic residues (H8-H12) were selectively replaced or important amino acid asparagine Asn repeated at positions 943 and 944 to further alter or optimize the polypeptide sequence (H10-H12).

The invention adopts a solid phase synthesis polypeptide technology to synthesize a linear polypeptide sequence, and then uses a method of closing a ring by using terminal aspartic acid to stabilize the alpha helical polypeptide of the target HRas.

Experiments prove that the polypeptide H5 has higher binding affinity with HRas, can selectively kill a cancer cell line with high Ras expression, H5 can inhibit a Ras/MAPK signal channel and reduce the phosphorylation level of kinases such as downstream signals ERK and the like, and H5 can also effectively inhibit the proliferation of cancer cells and improve the radiation sensitivity of cervical cancer cells in radiotherapy. Monomethyl Auristatin E (MMAE) is a potential radiation sensitizer for radiotherapy, can be used as a positive control in radiation sensitizer research of radiotherapy, and experiments show that the polypeptide H5 and the MMAE can synergistically enhance the apoptosis condition after being used in combination, block cells in the stage G0/G1, and have more remarkable curative effect than that of the polypeptide H5 or MMAE when being used alone in the radiation sensitizer research of radiotherapy. Experiments show that the polypeptide-based Ras inhibitor can be potentially applied to radiation sensitizer for radiotherapy, the polypeptide inhibitor expands the research of conformation constrained peptide in the field of radiotherapy, provides a thought for developing efficient Ras-targeted inhibitor, and the combined administration of the polypeptide and MMAE expands the potential application of combined treatment in the field of radiotherapy.

Compared with the prior art, the invention has remarkable technical progress. The invention proves that the polypeptide can be well combined with HRas protein and inhibit the growth of cell lines such as cervical cancer cells with high Ras expression through experiments such as fluorescence polarization detection, MTT experiment and the like. The polypeptide has the functions of inhibiting the growth of cell lines such as a Ras high-expression cervical cancer cell and the like and increasing the radiation sensitivity of the cervical cancer cell to X-rays, is beneficial to solving the problem of the resistance of the cancer cell to X-ray radiotherapy, and simultaneously widens the application range of conformation-constrained peptides.

Drawings

FIG. 1 the binding affinity of H2-FITC and H5-FITC polypeptides to HRas proteins was determined by fluorescence polarization.

FIG. 2 is a circular dichroism spectrum of polypeptides in purified water.

FIG. 3 is a flow cytometry experiment to determine the membrane penetration ability of FITC-modified polypeptides.

FIG. 4 is a confocal laser microscopy image showing the determination of the membrane penetration ability of FITC-labeled polypeptides H2 and H5 and the approximate localization of the polypeptides in HeLa cells to the cytoplasm or nucleus.

FIG. 5 shows the serum stability assay of the polypeptide L1H2H 5.

FIG. 6 shows the hemolytic activity of polypeptides H2 and H5.

FIG. 7 the killing ability of the polypeptide L1H2H5 on HeLa, A549, HepG2 and 293T cells was analyzed using the MTT assay.

FIG. 8 is a Control group containing DMSO only, wherein MTT assay was used to analyze the killing ability of HeLa cells by the combination of polypeptides H2, H5, MMAE and H5+ MMAE.

FIG. 9 is a flow chart of the induction of apoptosis in HeLa cells by the combination of polypeptides H2, H5, MMAE and H5+ MMAE.

FIG. 10 is a statistical analysis of the induction of apoptosis in HeLa cells by the combination of polypeptides H2, H5, MMAE and H5+ MMAE. The proportion of cell numbers in the regions of Q3 (representing early apoptotic cells) and Q2 (representing middle and late apoptotic cells) was counted, respectively. The apoptosis experiment was repeated at least three times, and the data were collectively used as a histogram to analyze the apoptosis induced by the combination of polypeptides H2, H5, MMAE and H5+ MMAE.

FIG. 11 shows that the combination of polypeptides H2, H5, MMAE and H5+ MMAE blocked HeLa cells in the G2/M phase of the cell cycle.

FIG. 12 is a statistical plot of the cell cycle distribution of polypeptides H2, H5, MMAE and H5+ MMAE in combination on HeLa cells. Compared with the control group, the polypeptide H2, H5, MMAE and the combination group of H5 and MMAE have increased cell proportion in the G2/M phase, which shows that each experimental group can block cells in the G2/M phase. Data are shown as mean ± sd, repeated at least three independent experiments. The standard deviation is shown as error bars in the figure.

FIG. 13 is a graph investigating the effect of polypeptides on the level of phosphorylation of downstream ERKs in the Ras-Raf-MEK-ERK signaling pathway. (A) Treating HeLa cells with combination of polypeptides H2, H5, MMAE and H5+ MMAE for 24 hours, adding 20ng/ml EGF for stimulation for 10min, extracting cell lysate for western blot experiment; (B) and (3) carrying out semi-quantitative analysis on the result of the western blot experiment, namely analyzing the relative content of pERK/GAPDH.

FIG. 14 shows the clone formation experiment of HeLa cells treated with the combination of polypeptides H2, H5, MMAE and H5+ MMAE after X-ray irradiation. (A) Cloning to form a survival rate curve; adding medicine into cells, incubating for 24h, irradiating with X-rays at different doses, digesting the cells, counting, re-planting the cells in a six-well plate, waiting for 9-11 days to allow the cells to grow clones, dyeing, air drying and counting; a 10% survival score line (SF 10%) is plotted in the figure, while Vehicle is the DMSO control group; (B) clone formation experiments the average lethal dose D0 value for each experimental group. Experimental data was performed by the two-tailed Student's t test using GraphPad Prism 6.0 software. Levels of significance are shown as P <0.05 and P < 0.01. Data are shown as mean ± standard deviation, which is shown as error bars in the figure.

FIG. 15 is a schematic diagram of a polypeptide modified by ring closure for inhibiting Ras-Sos interaction, blocking Ras-related signaling pathway, and increasing sensitivity to cancer cell radiotherapy.

FIG. 16 shows a polypeptide of the present invention H5H-WRR-cyclo (isoDFFDap) -IYLTNILKTQEGN-NH₂HPLC profile of (a).

FIG. 17 shows a polypeptide of the present invention H5H-WRR-cyclo (isoDFFDap) -IYLTNILKTQEGN-NH₂Mass spectrum of (2).

Detailed Description

The invention is further described below with reference to the accompanying drawings.

Example 1 preparation and isolation and purification of the polypeptide:

the core steps for preparing the above-described stabilized polypeptide, using solid phase synthesis to synthesize the polypeptide, are as follows (taking the H5 polypeptide as an example):

the specific operation steps are as follows:

(1) polypeptide solid phase synthesis: weighing Rink amide MBHA resin in a peptide connecting tube, removing Fmoc protective groups on the resin by using morpholine/N, N-Dimethylformamide (DMF) with the reagent volume ratio of 50% (v/v) in the peptide connecting tube, and removing the Fmoc protective groups on the resin by using N-Dimethylformamide (DMF)₂The reaction was carried out for 30min twice with blowing to expose free amino groups on the resin. Then washed 6-9 times alternately with solvents such as DMF solvent/Dichloromethane (DCM)/N-methylpyrrolidone (NMP). Then 5eq Fmoc-protected amino acid with naked carboxyl, 5eq HCTU, 10eq DIPEA were added in N₂The reaction was carried out under blowing for 1 h/time and 2 times. Then washed 6-9 times alternately with DMF/DCM/NMP, etc. The Fmoc protecting group on the amino acid was then removed with 50% morpholine/DMF for 30 min/time twice. Then 5eq Fmoc-protected amino acid with naked carboxyl, 5eq HCTU, 10eq DIPEA were added in N₂The reaction was carried out under blowing for 1 h/time and 2 times. The process is circulated until the polypeptide is grafted.

(2) Amino acids with Allyl and Alloc protecting groups such as isoD and Dap amino acids (commercially available products) were dissolved in 0.1eq of Pd (PPh) in anhydrous and oxygen-free DCM₃)₄And 4eq of 1, 3-dimethyl barbituric acid, for 2-3h to remove the Allyl and Alloc groups for the next ring closure reaction. -COOH and-NH on amino acid side chains on polypeptides₂The dehydration ring-closing reaction of (1) benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP)/1-hydroxybenzotriazole (HOBt)/N-methylmorpholine (NMM) was carried out in a reaction equivalent of 2 eq: 2 eq: 2.4eq of the reaction solution was reacted for 2 hours, 2 times. The polypeptide resin after the reaction is condensed with methanol for 10 minutes, and then N is used₂The resin was blow dried. With shear liquid TFA/TIS/H₂O(V_TFA/V_TIS/V_H2O95%: 2.5%: 2.5%) of the polypeptide, soaking the resin for 2h, and cutting the polypeptide from the resin to obtain a crude polypeptide product. With N₂Drying the shearing liquid by blowing, removing other impurities by precipitating with glacial ethyl ether 50%Acetonitrile dissolves the crude polypeptide product. The polypeptide crude product is separated and purified by using HPLC high performance liquid chromatography to obtain a pure polypeptide product, the molecular weight of a target product is identified by using a mass spectrum technology, the polypeptide fragment is formed by sequentially connecting amino acids, each amino acid is connected with 3-5 amino acids, the target molecular weight is detected once by using a mass spectrum, and the structure of the product, such as the amino acid, is finally determined. The purity of the polypeptide product is identified by analytical High Performance Liquid Chromatography (HPLC) and mass spectrometry, the purity of the polypeptide (named as H5 polypeptide) is about 96%, the HPLC spectrogram is shown in figure 16, and the mass spectrogram is shown in figure 17.

Example 2 Fluorescent Polarization (FP) assay screening for Polypeptides that bind to HRas

The binding affinity of FITC-labeled polypeptides to HRas proteins was determined using Fluorescence Polarization (FP) assay, and the results are summarized in Table 1. According to the results of the FP experiments, the linear polypeptide L1 only shows weak interaction with the HRas protein, indicating that the linear polypeptide L1 has weak affinity with the HRas, and can be used as a control polypeptide for subsequent experiments based on the original sequence designed from the key fragment of the Sos protein.

Polypeptides H3 and H4 designed based on the polypeptide sequence (FEGIYRLELLKAEEAN) have certain interaction with HRas, but the binding affinity is above micromolar level, and the binding affinity is weak, which indicates that an inhibitor designed based on the polypeptide sequence needs further optimization to have better effect, so that the sequence is not deeply researched subsequently. Notably, the loop-closing polypeptides H2 and H5, which were designed based on the original sequence of the Sos1 α H-helix (FFGIYLTNILKTEEGN), showed higher binding affinity to HRas proteins, K_DThe values are 528.5nM and 127.1nM respectively (FIG. 1), wherein the sequence of the polypeptide H5 is based on the sequence of the polypeptide H2, the second glutamic acid on the C terminal is mutated into glutamine, and the result shows that the mutation of a single amino acid on the polypeptide can cause obvious change of the binding affinity of the polypeptide and the target protein, thereby providing a thought for designing a reasonable polypeptide inhibitor. Subsequent studies focused on the biological application of the H5 polypeptide.

TABLE 1 sequences of Ras polypeptide inhibitors and binding affinities of the polypeptides to HRas

Example 3 circular dichroism results of Ras Polypeptides

In order to determine the secondary conformation of the polypeptide in an aqueous solution, the present invention determines the Circular Dichroism (CD) spectrum of the polypeptide by using a circular dichroism chromatograph. According to the characteristic negative absorption peaks near 208nm and 222nm and the characteristic positive absorption peak near 190nm in the circular dichroism chromatogram, the polypeptide can be judged to have a typical alpha helical conformation. The CD experimental results show that the polypeptides H2, H3, H5, H8, H10 and H11, which are ring-closed using the terminal aspartic acid nucleation template, show a typical alpha-helix conformation in their Circular Dichroism (CD) measurement experiments (fig. 2), while the linear polypeptide L1, which is not ring-closed, shows a random coil conformation, which confirms that the polypeptide can be stabilized in the alpha-helix conformation with greater efficiency using the TD terminal nucleation template.

Example 4 flow cytometry experiments to determine the transmembrane Capacity of Polypeptides

The cell membrane penetrating capacity of the polypeptide on cancer cells is an important factor for representing whether the polypeptide can play the function in the cells, and the cell membrane penetrating capacity of the polypeptide is discussed by performing a flow cytometry experiment and a laser confocal microscope imaging experiment.

The polypeptides were first Fluorescein Isothiocyanate (FITC) modified and HeLa cells were treated with 5 μ M FITC-labeled polypeptide for 1 hour, then washed with PBS and 0.4% trypan blue to remove excess FITC-labeled polypeptide, trypsinized cells, washed, and then analyzed by flow cytometry. Experimental results show that compared with the classical membrane-penetrating peptide TAT-FITC (the polypeptide sequence is FITC-RKKRRQRRR), the polypeptides H2-FITC and H5-FITC have better membrane-penetrating capability of cell membranes (figure 3), and other polypeptides show weaker membrane-penetrating capability of cell membranes compared with TAT-FITC. In flow cytometry experiments, TAT-FITC was used as a positive control and DMSO was used as a negative control.

Example 5 confocal laser microscopy imaging to investigate the membrane penetrating ability of the polypeptide

In order to further study the cell membrane penetrating capability of the polypeptide and the possible approximate location of the polypeptide in the cell, the invention carries out laser confocal microscope imaging experiments on the cell treated by the polypeptide. First, HeLa cell seeds were grown overnight in a 24-well plate containing cell circular slides, then HeLa cells were treated with 5 μ M FITC-labeled polypeptide for 1 hour, then washed alternately with PBS and 0.4% trypan blue to remove excess polypeptide attached to the cell membrane surface, then cell slides were mounted on slides with mounting medium containing DAPI dye (a dye that binds DNA efficiently and can be used to stain the nucleus), dried naturally, and finally imaged with a confocal laser microscope with the DAPI dye being excited with excitation light at 405nm channel and the FITC dye being excited with excitation light at 488nm channel. In a confocal laser microscopy imaging experiment, polypeptide TAT-FITC with good membrane penetrating capacity is used as a positive control, DMSO is used as a negative control, and the membrane penetrating capacity of cell membranes of the polypeptide is compared relatively. Confocal microscopy imaging results show that compared with a positive control cell-penetrating peptide TAT-FITC, cells treated by the polypeptides H2-FITC and H5-FITC can both find obvious green fluorescence in the cells, which indicates that the polypeptides H2-FITC and H5-FITC show stronger cell membrane penetrating capacity, and the polypeptides H2-FITC and H5-FITC are distributed in cytoplasm and nucleus (figure 4). The experimental result of confocal microscope imaging is consistent with the experimental result of the flow cytometry, and both show that the polypeptides H2-FITC and H5-FITC have stronger cell membrane penetrating capability and can penetrate through cell membranes to enter the cells, thereby better targeting the target protein in the cells to play the biological function of the target protein.

EXAMPLE 6 serum stability assay for Polypeptides

The experiments show that the H2 and H5 polypeptides have high binding affinity with HRas and good cell membrane penetrating capacity, so that the most representative H2 and H5 polypeptides are selected for further research in the following research. The stability of the polypeptide is an important factor for the polypeptide to exert its biological function in vivo, and then the stability of the polypeptide is characterized by an in vitro serum stability experiment. The results of the experiment show that approximately 60% of the polypeptides H2 and H5 remained intact and stable in 25% of mouse serum after the polypeptides were incubated with 25% of mouse serum in a water bath at 37 ℃ for 24 hours (FIG. 5), while the linear peptide L1 was rapidly degraded by the mouse serum, indicating that the alpha-helical polypeptide had higher stability in the serum after being subjected to ring closure by the N-terminal aspartic acid nucleation template.

Example 7 hemolytic assay of polypeptides

The hemolysis experiment of fresh mouse red blood cells is a research experiment for detecting hemolysis caused by the damage of polypeptide to cell membranes, and the premise that the polypeptide has lower hemolysis activity to the red blood cells ensures that the polypeptide can be safely used for cell experiments. In the hemolytic activity assay, Triton X100 is a class of nonionic surfactants, 0.1% Triton X100 can obviously destroy cell membranes, and is used as a positive control in hemolytic experiments. The results of the hemolytic experiments on the polypeptides show that the polypeptides H2 and H5 show lower hemolytic property at the concentration of the polypeptides lower than 40 mu M (figure 6), which indicates that the polypeptides have lower non-specific toxicity to cell membranes, and indicates that the polypeptides can be safely used for the next cell experiments.

Example 8 determination of cell viability by MTT assay

To further evaluate the killing effect of these polypeptides on cancer cells, an MTT assay cell viability assay was performed. The invention systematically studies the proliferation inhibition effect of the polypeptides H2 and H5 on Ras over-expressed cancer cell lines (HeLa, A549 or HepG2 cells) and common 293T cells, and the L1 linear polypeptide is used as a polypeptide control group. The MTT experimental results showed that the polypeptide H5 was able to inhibit proliferation of HeLa, a549 and HepG2 cells in a dose-dependent manner and showed lower toxicity to 293T cells, indicating that the polypeptide H5 had higher selectivity to Ras-overexpressed cancer cell lines (fig. 7); the H2 polypeptide also has certain killing capacity on HeLa, A549 and HepG2 cells, but has weaker effect than that of the H5 polypeptide; the H5, H2, and L1 linear polypeptides also have lower cytotoxicity to 293T cells.

H5 shows high selectivity and killing ability to Ras over-expressed cancer cell line, and then a more representative H5 polypeptide is used for research of drug combination with a potential radiosensitizer monomethyl auristatin E (MMAE). The killing ability of H2, H5 polypeptide, MMAE and H5+ MMAE in combination on HeLa cells was first tested (fig. 8). In the early experiments, when the use ratio of the combination of the drugs is explored, the MMAE has stronger killing capacity on cells, 5nM MMAE can kill most of the cells, and in order to reasonably research the effect of the combination of the polypeptide and the MMAE and reduce the use concentration of the MMAE, the experiments find that 0.5nM MMAE and 10 mu M H5 polypeptide can kill about 50% HeLa cells, so the use ratio of the combination is set to 10 mu M H5 polypeptide +0.5nM MMAE. The experimental result shows that the H2 and H5 polypeptides can obviously inhibit the proliferation of HeLa cells under the concentration of 10 mu M, and the result is consistent with the MTT experimental result; the MMAE has a relatively obvious effect of inhibiting the proliferation of the HeLa cells under the low concentration of 0.5nM, the H5 polypeptide and the MMAE show a stronger inhibition effect on the aspect of inhibiting the proliferation of the HeLa cells when the combination is used, and the curative effect of inhibiting the proliferation of the cancer cells by the synergistic effect of the H5 and MMAE combination is preliminarily shown.

Example 9 determination of apoptosis by Annexin-V/PI double staining assay

According to literature research, the Ras/MAPK signal channel is involved in the regulation of cell physiological activities such as cell growth and development, cell apoptosis and the like, and in order to evaluate whether the polypeptide has influence on the apoptosis phenomenon of HeLa cells, the invention carries out Annexin-V/PI double staining experiment to determine the apoptosis condition. The experimental result shows that the polypeptides H2 and H5 can induce HeLa cells to generate obvious apoptosis phenomenon, namely the proportion of the number of cells with early apoptosis and middle and late apoptosis represented by the Q3Q2 region in a flow cytometry is increased (figure 9). It is noted that the combination of the polypeptide H5(10 μ M) + MMAE (0.5nM) can enhance the effect of inducing HeLa cells to generate early apoptosis and middle and late apoptosis (fig. 10), and has stronger effect of inducing cells to generate apoptosis compared with the single polypeptide drug or single MMAE drug, indicating that the combination of drugs has synergistic effect of inducing apoptosis.

Example 10 cell cycle experiments

The present invention next investigated the effect of the polypeptide on the cell cycle of HeLa cells. In the cell cycle experimental assay, HeLa cells are firstly cultured in a six-well plate, then incubated with HeLa cells with specific concentrations of the combination of the polypeptides H2, H5, MMAE and H5+ MMAE for 24 hours, digested, centrifuged, collected, washed with PBS, fixed with 70% glacial ethanol for more than 4 hours, washed with PBS, then stained with propidium iodide PI in a water bath at 37 ℃ for 30 minutes, and finally the cell cycle arrest of HeLa cells is detected by a flow cytometer.

According to the change of the cell number ratio of G0/G1, S phase and G2/M phase of each experimental group relative to the DMSO control group in the flow cytometry, the cells can be judged to be blocked in a specific cell cycle. The experimental results show that the HeLa cells treated by the H2 polypeptide, the H5 polypeptide, the MMAE and the H5+ MMAE combination group show a significant increase in the ratio of the cell number in the G2/M phase relative to the control group (FIG. 11); statistical analysis is carried out according to the cell number proportion of each cell cycle, and the results show that the mitotic cell cycle of the cells of the H2 polypeptide, the H5 polypeptide, the MMAE and the H5+ MMAE combined drug group is blocked at the G2/M phase, wherein the H5+ MMAE combined drug group shows better effect than the drug group which is singly added, and the H5 polypeptide and the MMAE can possibly play a synergistic effect to block the cells at the G2/M phase (figure 12).

Example 11 Western blotting assay to determine the Effect of Polypeptides on the Ras Signal pathway

A large number of literature reports indicate that small molecule or polypeptide inhibitors inhibiting Ras proteins influence phosphorylation cascades downstream of Ras-Raf-MEK-ERK signaling pathways, and the phosphorylation levels of Raf, MEK and ERK proteins are reduced. The present invention uses western blot experiments to test the effect of polypeptides on the Ras-Raf-MEK-ERK signaling pathway, mainly investigating the effect on the phosphorylation level of the ERK protein most downstream of the signal. Incubating the polypeptide and HeLa cells for 24h, adding 20ng/ml growth factor (EGF) for stimulating for 10min, collecting culture medium supernatant cells and bottom cells, extracting cell lysate, and performing western blot experiment to determine the change of ERK protein phosphorylation level. The experimental results show that the combined administration group of H5 polypeptide, MMAE and H5+ MMAE can obviously inhibit the phosphorylation level of ERK protein (FIG. 13)

Example 12 clonogenic assay to determine the radiosensitivity of a polypeptide

The clone formation experiment can characterize the radiotherapy sensitization of cancer cells to drugs. Cell cloning, i.e. after attachment of single cellsProliferating and growing to form clones with a certain cell number; cells capable of forming clones are bound to adhere to the wall and have strong proliferation activity, and cells with weak proliferation activity after drug treatment and X-ray irradiation may not form clones. The invention adopts a clone formation experiment to test whether the polypeptide can cause the radiotherapy sensitivity of the HeLa cell to X-ray. MMAE is a reported potential radiosensitizer that can be used as a positive control for single-component dosing in this experiment. D₀The value is one of the important parameters for assessing radiosensitivity, D₀The value is the average lethal dose, theoretically the average radiation dose that results in each cell being hit by X-rays. The survival rate curve of the formed clone is obtained by fitting the survival rate data of the formed clone by using a single-click multi-target model, and the average lethal agent D of each experimental group can be directly obtained₀The value is obtained.

Fig. 14A shows the survival curves of clones from experimental groups after treatment of HeLa cells with the polypeptide and X-ray irradiation, and the survival curves of clones were significantly down-regulated by the polypeptide H5, MMAE and H5+ MMAE compared to Vehicle (DMSO control). H5 polypeptide and D of MMAE panel compared to Vehicle₀The values were significantly reduced (fig. 14B), indicating that both H5 polypeptide and MMAE can reduce clonogenic survival and increase radiation sensitivity of HeLa cells. Notably, the combination of 10 μ M H5 polypeptide with 0.5nM MMAE showed a smaller D₀The value shows that the H5+ MMAE combined drug can exert a synergistic effect to further enhance the radiotherapy sensitization, and fully shows the potential application of the H5+ MMAE combined drug in the aspect of radiotherapy sensitization research.

As shown in FIG. 15, based on a segment of alpha-helical polypeptide on the Sos protein on the interaction interface in the crystal structure of the Ras-Sos1 protein complex, the invention synthesizes the polypeptide after being subjected to the close-loop modification by the TD strategy, carries out site-directed amino acid mutation on the polypeptide, and screens an effective polypeptide inhibitor targeting the Ras protein, so as to inhibit the Ras-Sos interaction and further block the Ras/MAPK signal path, thereby increasing the radiotherapy sensitivity of cancer cells to X-rays. RTKs represent receptor tyrosine kinases.

Sequence listing

<110> Shenzhen institute of university of Beijing

Shenzhen Bay Laboratory Pingshan Biomedical R & D Conversion Center

<120> HRas protein targeted alpha helix polypeptide inhibitor and application thereof

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Claims (2)

1. A pharmaceutical composition, comprising an HRas protein-targeting alpha-helix polypeptide inhibitor and monomethyl auristatin, wherein the HRas protein-targeting alpha-helix polypeptide inhibitor has the following structural formula:

wherein isoD is L-isoaspartic acid, Dap represents 2, 3-diaminopropionic acid; the concentration of the alpha helix polypeptide inhibitor targeting the HRas protein is 10 mu M, and the concentration of the monomethyl auristatin E is 0.5 nM.

2. Use of the pharmaceutical composition of claim 1 in the manufacture of a medicament for increasing sensitivity to radiation therapy of cancer cells highly expressed by Ras.

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